Gold particle size will influence labeling density and selection
of instrument magnification when used in immunoelectron microscopy.
The tables and figures below should aid the researcher in choosing
the gold particle best suited to his or her research requirements
(reproduced with permission from Giberson and Demaree: The Influence
of Immunogold Particle Size on Labeling Density. Microscopy Research
and Technique 27:355-357, 1994) .

Notes: Gold conjugates 5nm to 30nm are
packaged with 20% glycerol and can be frozen. 1nm conjugates
are not packaged with glycerol and should not be
frozen. EM Gold conjugates - %CV: <12% for 5nm; <8% for
10,15 & 20nm; <20% for 30nm .
%Singlets: >85% for all conjugates except Protein A >95%.

These gold conjugates are made to the highest standards and specifications, yielding excellent
results when correctly used. The guidelines are straightforward and intended for
use with either light or electron microscopy
immunolabeling applications. All gold conjugates are supplied in one of the two buffers
listed below. Buffer constituents
dictate the shelf life and storage conditions for the individual conjugates.

Electron microscopy (EM) conjugates are supplied in the following buffer:
20mM Tris (tris-hydroxymethyl-aminomethane); 20mM sodium azide; 154mM NaCl; 20% glycerol;
pH 8.2. Recipe to make 100ml:
0.242g (20mM) Tris + 0.9g (154mM) NaCl + ultrapure water to make 100ml. Adjust pH
from 7.2 to 8.2 with 1N HCl or 1N NaOH.
Storage: Stable for 1 year at 4°C; stability for 2+ years at 20°C.
The conjugates demonstrate remarkable
stability at ambient temperatures for up to 7 days. Repeat freezing and thawing is not recommended.

Ultra small (2nm) gold conjugates are supplied in the following buffer: 10mM Na2HPO4;
3mM KH2PO4; 123mM NaCl; 1% glycerol; 20mM sodium azide; pH 7.2.
Recipe to make 100ml: 0.148g Na2HPO4 + 0.042g KH2PO4 + 0.72g NaCl + ultrapure water to make 100ml.
Adjust pH from 7.2 to 8.2 with 1N HCl or 1N NaOH. Storage: Stable for 1 year at 4°C - DO NOT FREEZE.
The conjugates demonstrate remarkable stability at ambient temperatures for up to 7 days.

Product Information
Each gold conjugate has a technical data sheet which indicates the following information:
1) Number of particles counted;
2) Mean particle diameter; 3) Coefficient of variation given as a percent; 4) Percent
of single particles; 5) Percent of
particles larger than triplets; and 6) Minimum detectable protein. The coefficient
of variation is an important parameter
in describing the relative distribution of gold particle sizes around the mean for
a given batch. The coefficient of variation
equals the standard deviation divided by the mean.
Normal Gaussian distributions work as follows: ±1 standard deviation describes
68% of the area under the curve; ±
2 standard deviations describe 95% of the area under the curve; ± 3 standard
deviations describe 99.73% of the area under
the curve. As an example, you have purchased a gold conjugate - Goat anti-Rabbit
IgG (H+L), 10nm - having a mean particle diameter
of 9.8nm with a coefficient of variation of 4.1%. First, the standard deviation needs
to be determined. In this case it is 0.402nm
(4.1% x mean particle diameter). Statistically, 68% of the particles will be from
9.40 to 10.20nm, 95% from 9.00 to 10.60nm and 99.73%
from 8.60 to 11.00nm. A reliable size characterization has been determined for the batch.

Sample Fixation
All antigens and tissue react differently to different fixatives and preservation
strategies (e.g. high pressure freezing,
cryoultramicrotomy, etc.). As a result no one method will produce results all the
time. Fixation is a very important step in
determining the success of most immunolabeling procedures. Paraformaldehyde-based
fixatives are most often used in concentrations
from 0.5% up to 4% or higher. Often glutaraldehyde is added in concentrations typically
from 0.1 to 0.5%. Cacodylate and the
phosphate (Millonig, Sörensen) buffers are popular and the zwitterionic buffers
such as HEPES or PIPES can be used as well
for aldeyde fixation. The quenching of unreacted aldehydes is recommended by one
of the following methods: 1) 0.05-0.1M ammonium
chloride in buffer at 4°C for a few hours; 2) 0.1-0.2M glycine in buffer for
a few hours; or 3) Sodium borohydride (0.1 mg/ml)
in buffer for a few hours. Osmium tetroxide is not often used for tissues processed
for immunolabeling. Berryman and Rodewald, 1990,
J. Histochem, Cytochem., 38: 159-170 describe an excellent method to attain membrane
contrast without the use of osmium.

Resin Infiltration/Polymerization
The hydrophilic polar resins (Lowicryl K4M and K11M; L R White; L R Gold) have
proven to be the best based on immunoreactivity.
Cryoultramicrotomy methods are more difficult but
yield excellent results when done correctly. The polar resins are partially water
soluble and do not need to be dehydrated to 100%.
Apolar hydrophobic resins (Lowicryl HM20 and HM23) are available for use as well.
The epoxy resins, for most applications, are not
recommended. Dehydration, infiltration and polymerization with polar and apolar resins
can be done at room temperatures or in the
cold (progressively lower temperature techniques). Polymerization of polar and apolar
resins is done by UV for immuno applications;
however, UV as well as oven cures can be done with LR White.

Positive/Negative Controls
Positive as well as negative controls should be a part of all immunolabeling studies.
Reagent and system dynamics require that
controls be used to identify optimum signal-to-noise ratios (i.e. correct dilution
of primary and secondary antibodies), viability
of antibodies being used and necessary blocking steps or strategies. Tween 20®
(Product 15713), Fish gelatin (45%)
(Product15717) are good generic blocking reagents that can be mixed
with antibody or rinse solutions.

Sample Labeling Protocol
Immunolabeling should be done in a humid chamber to protect against drying. The steps are:

5. Buffer Rinse: A method found beneficial to reduce background is to elevate the
NaCl concentration to approximately 2.5M
from 1.54mM. The change in molarity has a beneficial effect on background due to
ionic interaction of the gold conjugate (net negative
change) and the substrate. Rinse for approximately 10 minutes and switch to water
(ultrapure) for at least four changes of five minutes
each.

6. Post Stain: 2% uranyl acetate (aqueous or alcoholic) for 5 minutes, rinse well
in ultrapure water and then stain for 5 minutes
with lead (25ml DI water, dissolve one sodium hydroxide pellet and add 0.125g of
lead citrate). Rinse well after lead staining.

NOTE: Elevated temperatures (i.e. 37°C) during incubations require less time.
Less time helps to keep the sections on the grids.
Nickel or gold grids should be used. All grids, regardless, should be cleaned prior
to use (1N HCl; 100% ETOH; 100% Acetone - Dry in
60°C oven on filter paper).

Questions and Customer Support: If there are any questions,
please contact Ted Pella, Inc. and ask for Cindy Smith, Technical
Product Specialist.